Genomic patterns of DNA methylation in mammals play a critical role in gene regulation and chromatin organization. DNA methylation is important in a diverse range of cellular functions and pathologies, including tissue-specific gene expression, cell differentiation, genomic imprinting, X chromosome inactivation, regulation of chromatin structure, aging, and carcinogenesis. CG methylation is the most common type found in mammalian genomes and it is maintained by the maintenance DNA methyltransferase called DNMT1, which acts on newly synthesized DNA at replication forks and shows a preference for hemimethylated CG sites. UHRF1 contains a methyl DNA binding domain that shows strong preferential binding to hemimethylated CG sites. DNMT1 is recruited to replication foci in part through its interaction with proliferating cell nuclear antigen (PCNA), and in part by directly interacting with UHRF1. How methylated DNA is maintained at sites of gene silencing in normal and transformed cancer cells is still relatively unclear. UHRF1 binds hemimethylated DNA through its SRA domain;however, it is unclear what roles the various other domains of UHRF1 play in maintaining DNA methylation. Our preliminary data has revealed a previously unidentified site of methylation on DNMT1. We believe this might be another mechanism for regulating the maintenance DNA methylation. The goal of this project is to understand in greater detail how methylated DNA is maintained at sites of gene silencing in normal and transformed cancer cells. UHRF1 with mutations of the ubiquitin like domain, PHD, SRA, and the RING finger motif, respectively, will be stably expressed in murine embryonic stem (ES) cells with a targeted disruption of UHRF1. Various assays will be employed to help elucidate the roles these domains play in maintaining DNA methylation. Synthesized methylated peptides of DNMT1 will also be tested for direct binding to the UHRF1 PHD domain in vitro compared to unmethylated peptides. The function of methylated DNMT1 will also be examined by performing knockout and knockdown of the methyltransferase required for methylating DNMT1. Finally, an shRNA screen in murine ES cells will be developed to identify other genes required for DNA methylation. There is growing evidence that the DNA methylome undergoes characteristic changes in the majority of cancers examined thus far. This is highly relevant to mechanisms of carcinogenesis, since mis-regulated DNA methylation patterns can promote abnormal gene expression and metastatic properties of numerous cancer cell types. The long-term goal of these studies is to determine what role DNA methylation is playing in the transformation of a normal cell to a cancer phenotype. [unreadable]